Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression

Abscisic acid (ABA) is a plant hormone synthesized during seed development that is involved in the induction of seed dormancy. Delayed germination due to seed dormancy allows long-term seed survival in soil but is generally undesirable in crop species. Freshly harvested seeds of wild-type Nicotiana plumbaginifolia plants exhibit a clear primary dormancy that results in delayed germination, the degree of primary dormancy being influenced by environmental culture conditions of the mother plant. In contrast, seeds, obtained either from ABA-deficient mutant aba2-s1 plants directly or aba2-s1 plants grafted onto wild-type plant stocks, exhibited rapid germination under all conditions irrespective of the mother plant culture conditions. The ABA biosynthesis gene ABA2 of N. plumbaginifolia, encoding zeaxanthin epoxidase, was placed under the control of the constitutive 35S promoter. Transgenic plants overexpressing ABA2 mRNA exhibited delayed germination and increased ABA levels in mature seeds. Expression of an antisense ABA2 mRNA, however, resulted in rapid seed germination and in a reduction of ABA abundance in transgenic seeds. It appears possible, therefore, that seed dormancy can be controlled in this Nicotiana model species by the manipulation of ABA levels.     

Regulation of hormone metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism

In a wide range of plant species, seed germination is regulated antagonistically by two plant hormones, abscisic acid (ABA) and gibberellin (GA). In the present study, we have revealed that ABA metabolism (both biosynthesis and inactivation) was phytochrome-regulated in an opposite fashion to GA metabolism during photoreversible seed germination in Arabidopsis. Endogenous ABA levels were decreased by irradiation with a red (R) light pulse in dark-imbibed seeds pre-treated with a far-red (FR) light pulse, and the reduction in ABA levels in response to R light was inhibited in a phytochrome B (PHYB)-deficient mutant. Expression of an ABA biosynthesis gene, AtNCED6, and the inactivation gene, CYP707A2, was regulated in a photoreversible manner, suggesting a key role for the genes in PHYB-mediated regulation of ABA metabolism. Abscisic acid-deficient mutants such as nced6-1, aba2-2 and aao3-4 exhibited an enhanced ability to germinate relative to wild type when imbibed in the dark after irradiation with an FR light pulse. In addition, the ability to synthesize GA was improved in the aba2-2 mutant compared with wild type during dark-imbibition after an FR light pulse. Activation of GA biosynthesis in the aba2-2 mutant was also observed during seed development. These data indicate that ABA is involved in the suppression of GA biosynthesis in both imbibed and developing seeds. Spatial expression patterns of the AtABA2 and AAO3 genes, responsible for last two steps of ABA biosynthesis, were distinct from that of the GA biosynthesis gene, AtGA3ox2, in both imbibed and developing seeds, suggesting that biosynthesis of ABA and GA in seeds occurs in different cell types.     

The ABA1 gene and carotenoid biosynthesis are required for late skotomorphogenic growth in Arabidopsis thaliana

Several plant hormones, including auxin, brassinosteroids and gibberellins, are required for skotomorphogenesis, which is the etiolated growth that seedlings undergo in the absence of light. To examine the growth of abscisic acid (ABA)-deficient mutants in the dark, we analysed several aba1 loss-of-function alleles, which are deficient in zeaxanthin epoxidase. The aba1 mutants displayed a partially de-etiolated phenotype, including reduced hypocotyl growth, cotyledon expansion and the development of true leaves, during late skotomorphogenic growth. In contrast, only small differences in hypocotyl growth were found between wild-type seedlings and ABA-deficient mutants impaired in subsequent steps of the pathway, namely nced3, aba2, aba3 and aao3. Interestingly, phenocopies of the partially de-etiolated phenotype of the aba1 mutants were obtained when wild-type seedlings were dark-grown on medium supplemented with fluridone, an inhibitor of phytoene desaturase, and hence, of carotenoid biosynthesis. ABA supplementation did not restore the normal skotomorphogenic growth of aba1 mutants or fluridone-treated wild-type plants, suggesting a direct inhibitory effect of fluridone on carotenoid biosynthesis. In addition, aba1 mutants showed impaired production of the beta-carotene-derived xanthophylls, neoxanthin, violaxanthin and antheraxanthin. Because fluridone treatment of wild-type plants phenocopied the phenotype of dark-grown aba1 mutants, impaired carotenoid biosynthesis in aba1 mutants is probably responsible for the observed skotomorphogenic phenotype. Thus, ABA1 is required for skotomorphogenic growth, and beta-carotene-derived xanthophylls are putative regulators of skotomorphogenesis.     

The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers

A novel abscisic acid (ABA)-deficient mutant, aba4, was identified in a screen for paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds. Carotenoid composition analysis demonstrated that the defective locus affects neoxanthin synthesis. The ABA4 gene was identified by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has four putative helical transmembrane domains and shows significant similarity to predicted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans-neoxanthin, indicating that the ABA4 protein has a direct role in neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA-deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both violaxanthin and neoxanthin are in vivo substrates for 9-cis-epoxycarotenoid dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via neoxanthin isomer precursors.     

Abscisic-acid-deficient mutants at the aba gene locus of Arabidopsis thaliana are impaired in the epoxidation of zeaxanthin

Abstract. The xanthophyll content of wild type and abscisic acid (ABA) - deficient mutants of pea and Arabidopsis thaliana was determined. The wilty mutant of pea was indistinguishable from the non-mutant control. In contrast, plants homozygous for mutant alleles at the aba locus of Arabidopsis were very different from wild type. In these mutants, zeaxanthin accumulated to abnormally high levels. The major carotenoids, violaxanthin and 9'- cis -neoxanthin were virually absent from the mutant chromatograms. It was concluded that the aba genetic lesion impairs the epoxidation of zeaxanthin to violaxanthin and that this results in an inability to accumulate ABA. This provides clear evidence that zeaxanthin is a precursor of ABA.     

Analysis of phytochrome- and ABA-deficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia

The phytochrome chromophore-deficient mutant, pew1, of Nicotiana plumbaginifolia exhibited decreased germination and slower dehydration of detached leaves during water stress as compared with the wild-type. These physiological processes are controlled by abscisic acid (ABA) and we examined, therefore, whether phytochrome plays a specific role in the regulation of ABA metabolism using the pew1 mutant. The ABA contents of mature seeds and young leaves were analysed and in both cases mutant material was found to contain higher amounts of ABA as compared with the wild-type. This indicates that the phytochrome activation can lead to a decrease of the ABA level in the wild-type plant. The role of phytochromes was investigated in greater detail using the ABA-deficient mutant aba1 of N. plumbaginifolia exhibiting an early and synchronous germination. This mutant accumulates at very high levels a metabolite derived from a precursor (ABA-aldehyde) in the ABA biosynthetic pathway. The first biochemical characterization of this molecule, which corresponds to the glucose-conjugated ABA-alcohol (ABA-AG) is described. A pew1-aba1 double mutant exhibiting both an etiolated growth and early germination was also obtained. The comparable accumulation of ABA-AG in the pew1-aba1 double mutant as compared with the aba1 mutant allowed the proposition that, in a wild-type plant, the phytochrome-mediated light signal enhances ABA degradation rather than inhibits its biosynthesis.     

Involvement of endogenous abscisic acid in methyl jasmonate-induced stomatal closure in Arabidopsis

In this study, we examined the involvement of endogenous abscisic acid (ABA) in methyl jasmonate (MeJA)-induced stomatal closure using an inhibitor of ABA biosynthesis, fluridon (FLU), and an ABA-deficient Arabidopsis (Arabidopsis thaliana) mutant, aba2-2. We found that pretreatment with FLU inhibited MeJA-induced stomatal closure but not ABA-induced stomatal closure in wild-type plants. The aba2-2 mutation impaired MeJA-induced stomatal closure but not ABA-induced stomatal closure. We also investigated the effects of FLU and the aba2-2 mutation on cytosolic free calcium concentration ([Ca(2+)](cyt)) in guard cells using a Ca(2+)-reporter fluorescent protein, Yellow Cameleon 3.6. In wild-type guard cells, FLU inhibited MeJA-induced [Ca(2+)](cyt) elevation but not ABA-induced [Ca(2+)](cyt) elevation. The aba2-2 mutation did not affect ABA-elicited [Ca(2+)](cyt) elevation but suppressed MeJA-induced [Ca(2+)](cyt) elevation. We also tested the effects of the aba2-2 mutation and FLU on the expression of MeJA-inducible VEGETATIVE STORAGE PROTEIN1 (VSP1). In the aba2-2 mutant, MeJA did not induce VSP1 expression. In wild-type leaves, FLU inhibited MeJA-induced VSP1 expression. Pretreatment with ABA at 0.1 μm, which is not enough concentration to evoke ABA responses in the wild type, rescued the observed phenotypes of the aba2-2 mutant. Finally, we found that in wild-type leaves, MeJA stimulates the expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE3, which encodes a crucial enzyme in ABA biosynthesis. These results suggest that endogenous ABA could be involved in MeJA signal transduction and lead to stomatal closure in Arabidopsis guard cells.     

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.     

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.     

Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci

Novel Arabidopsis mutants with lowered levels of endogenous abscisic acid (ABA) were isolated. These were selected in a screen for germination in the presence of the gibberellin biosynthesis inhibitor paclobutrazol. Another mutant was isolated in a screen for NaCl tolerance. The ABA-deficiency was caused by two monogenic, recessive mutations, aba2 and aba3 , that were both located on chromosome 1. The mutants showed a phenotype that is known to be characteristic for ABA-deficiency: a reduced seed dormancy and excessive water loss, leading to a wilty phenotype. Double mutant analysis, combining different aba mutations, indicated the leaky nature of the mutations.     

The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde

Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobre?o (sa?). The sre and sa? mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sa?3-1, and sa?3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K(m) value for xanthoxin of 19 micro M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway.     

Comprehensive hormone profiling in developing Arabidopsis seeds: examination of the site of ABA biosynthesis, ABA transport and hormone interactions

ABA plays important roles in many aspects of seed development, including accumulation of storage compounds, acquisition of desiccation tolerance, induction of seed dormancy and suppression of precocious germination. Quantification of ABA in the F(1) and F(2) populations originated from crosses between the wild type and an ABA-deficient mutant aba2-2 demonstrated that ABA was synthesized in both maternal and zygotic tissues during seed development. In the absence of zygotic ABA, ABA synthesized in maternal tissues was translocated into the embryos and partially induced seed dormancy. We also analyzed the levels of ABA metabolites, gibberellins, IAA, cytokinins, jasmonates and salicylic acid (SA) in the developing seeds of the wild type and aba2-2. ABA metabolites accumulated differentially in the silique and seed tissues during development. Endogenous levels of SA were elevated in aba2-2 in the later developmental stages, whereas that of IAA was reduced compared with the wild type. These data suggest that ABA metabolism depends on developmental stages and tissues, and that ABA interacts with other hormones to regulate seed developmental processes.     

Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Zeaxanthin epoxldase (ZE) catalyses two early steps in the abscisicacid (ABA) biosynthetic pathway. The sequence of a cDNA cloneencoding ZE from Nicotiana plumbaginifolia was reported In 1996and represented the first DNA sequence data on an ABA biosyntheticenzyme. The N. plumbaginifolia cDNA has been used to providea heterologous probe to isolate a ZE cDNA from tomato (Lycopersiconesculentum Mill.). DNA and amino acid sequence differences areconsidered in relation to putative functional domans withinthe enzyme. The results of northern analysis in tomato are discussedin relation to the effects of water stress on ZE mRNA levels. Key words: ABA biosynthesis, zeaxanthin epoxidase, tomato     

Ectopic expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis promotes seed dormancy and stress tolerance

Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mm NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2::beta-glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress.     

高等植物脱落酸生物合成途径及其酶调控

脱落酸（ABA）生物合成一般有两条途径：C15直接途径和C40间接途径, 前者经C15法呢焦磷酸（FPP）直接形成ABA;后者经由类胡萝卜素的氧化裂解间接形成ABA, 是高等植物ABA生物合成的主要途径。9-顺式环氧类胡萝卜素氧化裂解为黄质醛是植物ABA生物合成的关键步骤, 然后黄质醛被氧化形成一种酮, 该过程需NAD为辅因子, 酮再转变形成ABA-醛, ABA-醛氧化最终形成ABA。在该途径中,玉米黄质环氧化酶（ZEP）、9-顺式环氧类胡萝卜素双加氧酶（NCED）和醛氧化酶（AO）可能起重要作用。